Evolutionary Insights into the Microneme-Secreted, Chitinase-Containing High-Molecular-Weight Protein Complexes Involved in Plasmodium Invasion of the Mosquito Midgut.

While general mechanisms by which Plasmodium ookinetes invade the mosquito midgut have been studied, details regarding the interface of the ookinete, specifically its barriers to invasion, such as the proteolytic milieu, the chitin-containing, protein cross-linked peritrophic matrix, and the midgut epithelium, remain to be understood. Here, we review our knowledge of Plasmodium chitinases and the mechanisms by which they mediate ookinetes crossing the peritrophic matrix. The integration of new genomic insights into previous findings advances our understanding of Plasmodium evolution. Recently obtained Plasmodium species genomic data enable identification of the conserved residues in the experimentally demonstrated hetero-multimeric, high-molecular-weight complex comprised of a short chitinase covalently linked to binding partners, von Willebrand factor A domain-related protein (WARP) and secreted ookinete adhesive protein (SOAP). Artificial intelligence-based high-resolution structural modeling using the DeepMind AlphaFold algorithm yielded highly informative three-dimensional structures and insights into how short chitinases, WARP, and SOAP may interact at the atomic level to form the ookinete-secreted peritrophic matrix invasion complex. Elucidating the significance of the divergence of ookinete-secreted micronemal proteins among Plasmodium species may lead to a better understanding of the ookinete invasion machinery and the coevolution of Plasmodium-mosquito interactions.

Structure-function analysis of cysteine residues in the plasmodium falciparum chitinase, PfCHT1.

The Plasmodium ookinete uses chitinase activity to penetrate the acellular, chitin-containing peritrophic matrix to invade the mosquito vector. Plasmodium ookinetes from different parasite clades secrete two structurally distinct forms of chitinase, one, a short form lacking a C-terminal putative chitin-binding domain (CBD), the other, a long form with both proenzyme and C-terminal putative chitin-binding domains. Here, we structurally and functionally characterize the three cysteines in the short chitinase of the human-infecting malaria parasite, P. falciparum testing the hypothesis that one unpaired cysteine would not contribute to chitinase-specific enzymatic activity which would identify this residue as potentially involved in intermolecular disulfide bonding and heteromultimeric invasion complex formation as previously described. To test this hypothesis, we produced and characterized recombinant wild-type and cysteine-mutation PfCHT1 proteins in E. coli and used biophysical and enzymatic approaches to examine their enzymatic activities and chitin-binding affinities. The cysteine-203 PfCHT1 mutation had no effect on chitinolytic and chitin-binding functions. The cysteine-220 and cysteine-230 mutants were enzymatically inactive and did not bind to chitin. The artificial intelligence-based protein prediction algorithm, AlphaFold, correctly identified the involvement of cys-220 and cys-230 in the intramolecular disulfide linkages key to maintaining properly folded chitinase structural integrity. AlphaFold predicted that cys-203 cysteine is surface exposed and thus involved in intermolecular protein-protein interaction. Production of the cys-to-ser 203 PfCHT1 mutant facilitated recombinant protein production. Future cellular and biochemical studies are needed to further understand details of Plasmodium ookinete mosquito midgut invasion.

Transmission-Blocking Preventive Measures for Infectious Diseases: Altruism, Solidarity, and the Common Good.

Aiming to prevent the spread of contagious diseases has long been a central tenet of public health. In the present time, divisive political responses to mask wearing to prevent SARS-CoV-2 transmission have competed with sound public health advice for public attention. Here, we draw parallels in terms of individualism versus societal solidarity between the slow and ponderous development of transmission-blocking vaccines for malaria and advocacy for mask wearing to prevent COVID-19.